The present invention is directed to arrays for identifying large numbers of biomolecules in a biological sample so as to help determine their function and role in disease. More particularly, the invention relates to arrays of membranes for detecting and identifying large numbers of biomolecules in a multiplex manner. The application is a continuation-in-part of PCT application US00/20354 entitled Method and Production of Layered Expression Scans For Tissue and Cell Samples, which is incorporated herein by reference.
Now that the 100,000 or so genes that make up the human genome have been sequenced, new tools are needed to determine when and in what type of tissue those genes are active so as to ascertain their function and role in disease. This effort, often referred to as xe2x80x9cfunctional genomicsxe2x80x9d and xe2x80x9cproteomics,xe2x80x9d is especially important in efforts to discover new drugs since most new pharmaceutical agents are being designed to target enzymes, receptors, and other proteins. Eventually, this information will be used in clinical diagnostics to help guide treatment selection in the emerging era of xe2x80x9cpersonalized medicine.xe2x80x9d
Some believe that the 100,000 human genes may turn out to produce up to a million different protein variants. Of these, it is estimated that about 10,000 proteins will be identified over the next ten years as targets of pharmaceutical intervention. However, only a small fraction of these proteins are expressed in any particular tissue type. For example, a very different set of genes is expressed in brain tissue from those expressed in kidney even though cells in both organs have the same set of genes. Moreover, the subset of genes expressed in a kidney tumor differ from those active in healthy tissue from that organ. It is clear, therefore, that tools are needed to identify the activity of large numbers of genes in tissue samples removed from subjects.
To meet this need a number of xe2x80x9cmultiplexxe2x80x9d assays have been introduced.
Among the most common type of assays for surveying the expression of large numbers of genes in parallel are DNA microarrays (a/k/a xe2x80x9cbiochipsxe2x80x9d). Most microarrays consist of a glass slide or other solid surface upon which thousands of cDNA probes are anchored. With these devices DNA probes are arrayed in a grid-like format. Messenger RNAs are isolated from the samples of interest and allowed to hybridize to the probes anchored to the biochip revealing the profile of the genes expressed. Various scanners and software programs are used to profile the patterns of genes that are xe2x80x9cturned on.xe2x80x9d Representative of this biochip approach is the GeneChip(copyright) system from Affymetrix, Inc. (Santa Clara, Calif.).
While there are many uses for the aforementioned DNA microarrays, there are several limitations. First, they do not detect proteins, only nucleic acids. Since mRNA and protein levels do not always correlate in the cell and many regulatory processes occur after transcription, a direct measure of proteins is more desirable. Thus, since mRNA and protein levels do not always correlate in the cell and many functional protein modifications occur after translation, a direct way to monitor proteins is needed.
Another disadvantage of the microarrays known in the art is the fact that the sample being tested is disassociated from the tissue from which it was isolated. Disease is the result of disturbed biological equilibrium in groups of cells. Thus, it is often desirable to observe gene expression patterns in the context of the tissue in which the genes are active. In situ detection and visualization of proteins traditionally has been accomplished through immuno-histochemistry (IHC). This technique involves mounting a thin tissue section on the glass slide and visualizing a protein of interest with a detectable antibody that has specific binding affinity for the target protein. Because of certain technical limitations of IHC, only one or two proteins from a single tissue section can be analyzed. Also, proteins are still embedded in the tissue and are not presented to the antibodies in the most appropriate way (proteins are not highly denatured) lowering the success rate of the antibody reactivity.
Additionally microarrays known in the art require that in order to collect enough of the material for analysis, the sample being tested be a mixture of a number of different cell types (diseased tissue and adjacent normal cells) that are disassociated together and used for biomolecule extraction. As the result of this approach, biomolecules originating in the diseased tissue (e.g. tumor) are diluted and harder to detect and characterize. Since the morphological relationship is not preserved, it is hard to know what component of the sample is responsible for the changes observed in gene expression.
It is therefore desirable to have a method and device that combines the morphological advantages of IHC and other in-situ approaches with the multiplex and high-throughput characteristics of DNA microarrays.
To meet this need, Englert, et al. describe a very innovative technique which they refer to as xe2x80x9clayered expression scanningxe2x80x9d for molecular analysis of tumor samples that uses a layered array of capture membranes coupled to antibodies or DNA sequences to perform multiplex protein or mRNA analysis. Cancer Research 60, 1526-1530, Mar. 15, 2000. With this technique cell or tissue samples are transferred through a series of individual capture layers, each linked to a separate antibody or DNA sequence. As the biomolecules traverse the membrane set, each targeted protein or mRNA is specifically captured by the layer containing the corresponding antibody or cDNA sequence. The two-dimensional relationship of the cell populations is maintained during the transfer process thereby producing a molecular profile of each cell type present.
It would be desirable to supplement and enhance the layered expression scanning technique described by Englert, et al. with an approach that utilizes a stack of xe2x80x9cblankxe2x80x9d membranes that are not specific for any particular target. Instead, such membranes would ubiquitously bind to all (or a subset) of the biomolecules in a sample so as to give the user the flexibility of detecting a wide variety of biomolecules in an open format.
The present invention is directed to a device and a method for detecting biomolecules in a tissue section or other two-dimensional sample by creating xe2x80x9ccarbon copiesxe2x80x9d of the biomolecules eluted from the sample and visualizing the biomolecules on the copies using detectors, for example antibodies or DNA probes, having specific affinity for the biomolecules of interest.
Thin membranes in a stacked or layered configuration are applied to the sample, such as a tissue section, and reagents and reaction conditions are provided so that the biomolecules are eluted from the sample and transferred onto each of the stacked membranes thereby producing multiple substantial replicas of the biomolecular content of the sample. The treated membranes (or layers) are then separated. Each membrane is incubated with one or more different detectors (for example antibodies) specific for a particular biomolecule (such as a protein) of interest. The detectors employed are labeled or otherwise detectable using any of a variety of techniques such as chemiluminescence.
In an example in which proteins are detected, each membrane has essentially the same pattern of proteins bound to it, but different combinations of proteins are made visible on each membrane due to the particular antibodies selected to be applied. For example, one membrane layer may display proteins involved in programmed cell death (apoptosis) while an adjacent layer may display enzymes involved in cell division such as tyrosine kinases. In addition to proteins, nucleic acids may be targeted by using labeled DNA probes in lieu of antibodies. Moreover, different types of target biomolecules may be detected in different layers. For example, both protein and nucleic acid targets can be detected in parallel by applying both antibodies and probes to different layers of the array.
Another feature of the present invention is providing a kit that includes a group of membranes in a stack or other configuration that permits them to be stacked, and different detectors, such as cocktails of antibodies or probes, to be applied to the treated membranes for biomolecule detection.
With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the invention, the appended claims and to the several views illustrated in the drawings.